Organic vegetables are gaining popularity among customers due to the general belief that organic food is healthier and safer than vegetables grown with chemical fertilisers. The increased demand for organic vegetables prompted many small-scale farms to practice organic farming. In addition, many amateur and urban farmers also practised organic farming as a hobby. ¹

To fertilise farm soil with exogenous carbon and nitrogen at optimal carbon-to-nitrogen ratio ² and to reduce pathogen loads, ³ organic composts are typically prepared by composting nitrogen- and carbon-rich organic materials. ⁴ Typical raw materials for preparing organic composts include animal manures, protein-rich vegetation, and carbon-rich vegetation. For example, Meng et al., demonstrated that co-composting pig manure with spent mushroom substrate and rice husks increased the maturity and nutritional content of the compost product ⁵ and Gurtler et al., reported that composting manure effectively reduced the quantities of pathogenic microorganisms in organic composts. ⁶

In order to ensure that there is no carry-over of chemicals through organic composts and to standardise the quality of the produces, a stringent set of criteria on the origins and quality of these raw materials are in place in many organic farms of industrial scale or accredited organic farms under the National Organic Plan of the US ⁷ or other accreditation schemes. However, instead of following these stringent criteria, many farmers from small-scale organic farms or amateur and urban farmers often prepare their organic composts with raw materials that are easily accessible, such as animal manure in the farms, food wastes, and green waste, in order to reduce the production cost. In addition, standardisation in quality is not a significant concern for these farmers since many of their produces were sold to a small and specific group of customers or for personal consumption. ⁸

Although the use of organic compost can prevent the chemical hazards associated with chemical fertilisers and pesticides, organic composts in organic farming may increase the risk of disseminating antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). ⁹ For example, Lam et al., found a larger number of antibiotic-resistant bacteria and higher minimal inhibitory concentration (MIC) in organic lettuce sold in Hong Kong compared with the lettuce grown with chemical fertiliser and hydroponic methods. ¹⁰ It is thus evident that antimicrobial resistance (AMR) contamination in organic compost can pose a public health risk and warrant further investigation and surveillance.

Animal manure is generally believed to be the primary source of these antibiotic-resistant bacteria and ARGs. ¹¹ This belief originates from the fact that antibiotics are frequently used for promoting growth and curing veterinary diseases, ¹² and antibiotic-resistant bacteria are being selected and tainting animal manure. For example, Pan et al., analysed swine manure samples from 21 concentrated animal feeding operations in China and found that the detection frequencies of tetracyclines, sulfonamides and macrolides were 84.9–96.8%, 84.9–96.8% and 4.8%, respectively. ¹³ The presence of bacteria carrying antibiotic-resistant genes (ARGs) has been widely observed, and manure has been identified as the primary source of ARGs in organic vegetables. ¹⁴

Although antibiotic-resistant bacteria and resistome in various types of animal manure have been well characterised, few studies explored ARGs in other raw materials of organic composts. The contribution of these raw materials to the AMR risk of organic composts remains to be explored. Here we conducted a preliminary study to measure the ARG abundance and richness (the number of ARG subtypes) in three raw materials commonly used for preparing organic composts in small-scale organic farms in Hong Kong, which are chicken manure (CF), spent coffee grounds (COFFEE), and plant litter (chopped grass, GRASS). Surprisingly, our result indicated that, among the three raw materials, COFFEE harboured the highest ARG abundance with genes conferring resistance to multidrug being the predominating ARG subtypes. Our data challenged the general belief that animal manure is the primary source of ARGs in organic composts and suggested that other raw materials used for preparing organic compost may have a major contribution to the AMR risk of organic composts.

ARGs in CF, COFFEE, and GRASS samples were quantified using resistome analysis pipeline ARGs-OAP v2.0. A total of 21 ARG types and 420 subtypes were identified. Notably, the result indicated that COFFEE had the highest abundance of ARGs (average abundance 3.20 copies/cell), followed by CF (average abundance 2.20 copies/cell) and grass (average abundance 0.519 copies/cell) ( Figure 1a and Table 1). The total ARG abundances were significantly different between any of the two raw manure types (Tukey's HSD, adjusted p = 0.017, 0.001, and 9.76 x 10 ^-5 for CF vs. COFFEE, CF vs. GRASS, and COFFEE vs. GRASS, respectively; Figure 1b). In addition, multi-drug ARGs were most abundant in COFFEE (average abundance 2.4 copies/cell) compared to the other two raw material types. Aminoglycoside, MLS, sulfonamide, tetracycline, and chloramphenicol were the predominant ARG types in CF, where they together accounted for 85.1% of the total ARG abundance. The ARG alpha diversity represented by the total number of ARG subtypes ( i.e., richness) varied from 150 to 229 across all samples ( Table 1), with CF having a higher diversity than the COFFEE group (Tukey's HSD, adjusted p = 0.008, Figure 1c).

The demand for organic composts is increasing as organic farming is gaining popularity. Organic composts are often made of GRASS as the carbon sources and animal manure and other protein-rich materials, such as COFFEE, as the nitrogen sources. Since antibiotics have been frequently used in animal farming, residual antibiotics and ARGs are commonly found in animal manures. Thus, animal manure is generally believed to be the primary source of ARGs found in organic composts. However, there is little concrete evidence to support this belief. Therefore, this study examined this idea by characterising the resistome of the raw materials commonly used to produce organic composts in Hong Kong. To our surprise, instead of the animal manure, COFFEE carried the highest copy number of ARGs among the three raw materials used, and multi-drug resistance genes predominated its resistome. The abundance of multi-drug resistance genes in COFFEE was also the highest among the three raw materials. The high abundance of ARGs in COFFEE may be due to the infestation of fungi in COFFEE. These results challenged the general belief and highlighted the potential role of coffee grounds and possibly other plant-based food wastes in AMR dissemination. It is worth noting that a recent study also reported that corn stalk residue might add antibiotic-resistant bacteria to organic compost. ¹⁹ The high number of ARGs in COFFEE also leads to the speculation about whether ARG contamination is common in coffee, coffee beans and other coffee products. Further investigations are needed to confirm this study's findings, gauge the prevalence of AMR contamination in coffee and coffee products, and evaluate the role of food waste and green waste in contributing to the dissemination of AMR along the food chain and in the environment.